1,1 '-[[(substituted alkyl)imino]bis(alkylene)]bis-ferrocenes and their use in i electrochemical assays by labelling substrates of interest

Abstract

Compounds of general formula I wherein Fc and Fc′ may be the same or different and are substituted ferrocenyl moieties having at least one ring substituent selected from sulfur-containing groups, phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl groups containing two or more fluorine atoms, heteroaryl, substituted phenyl, and cyano, wherein if present as sole substituent the cyano group is located on the proximal cyclopentadienyl ring; X is a spacer, Y is a spacer, Z is a spacer; and R is a linker group. Compound I may be used to make labelled substrates, functionalised compounds for making labelled substrates and may be used as labels in an electrochemical assay.

1 . A method of detecting a substrate in an electrochemical assay comprising contact the substrate with a label comprising a compound of general formula I: 
       
         
           
           
               
               
           
         
       
       in which:
 Fc is a substituted ferrocenyl moiety having at least one ring substituent selected from sulfur-containing groups, phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl groups containing two or more fluorine atoms, heteroaryl, substituted phenyl, and cyano, wherein if present as sole substituent the cyano group is located on the proximal cyclopentadienyl ring; 
 Fc′ is a substituted ferrocenyl moiety having at least one ring substituent selected from sulfur-containing groups, phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl groups containing two or more fluorine atoms, heteroaryl, substituted phenyl, and cyano, wherein if present as sole substituent the cyano group is located on the proximal cyclopentadienyl ring, and may be the same as or different from Fc; 
 X is a spacer; 
 Y is a spacer; 
 Z is a spacer; 
 
       and
 R is a linker group. 
 
     
     
         2 . The method according to  claim 1 , wherein: X represents C1 to C6-alkylene optionally interrupted by oxygen; Y represents C1 to C6-alkylene optionally interrupted by oxygen, and Z represents C1 to C8 alkylene optionally interrupted by oxygen. 
     
     
         3 . The method according to  claim 1 , wherein: X is —(CH 2 ) x — in which x is 1 or 2; Y is —(CH 2 ) y — in which y is 1 or 2; Z is (CH 2 ) z  in which z is from 1 to 8. 
     
     
         4 . The method according to any  claim 1 , wherein Fc and Fc′ are the same and X and Y are the same. 
     
     
         5 . (canceled) 
     
     
         6 . The method according to  claim 1 , wherein the assay is for detecting a biological or synthetic substrate selected from nucleotides, nucleosides, oligonucleotides, and polynucleotides or from amino acids, peptides, and proteins. 
     
     
         7 . (canceled) 
     
     
         8 . A method for manufacturing a functionalized labelling compound comprising a label moiety for use in an electrochemical assay, comprising reacting a compound of general formula I 
       
         
           
           
               
               
           
         
       
       in which:
 Fc is a substituted ferrocenyl moiety having at least one ring substituent selected from sulfur-containing groups, phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl groups containing two or more fluorine atoms, heteroaryl, substituted phenyl, and cyano, wherein if present as sole substituent the cyano group is located on the proximal cyclopentadienyl ring; 
 Fc′ is a substituted ferrocenyl moiety having at least one ring substituent selected from sulfur-containing groups, phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl groups containing two or more fluorine atoms, heteroaryl, substituted phenyl, and cyano, wherein if present as sole substituent the cyano group is located on the proximal cyclopentadienyl ring, and may be the same as or different from Fc; 
 X is a spacer; 
 Y is a spacer; 
 Z is a spacer; 
 
       and
 R is a linker group, 
 
       with a functionalising compound to obtain a funtionalised labelling compound of general formula III:
   A-L-F  III
 
 
       in which A represents 
       
         
           
           
               
               
           
         
       
       wherein Fc, Fc′, X, Y and Z are as defined 
       above, 
       F represents a functionalising moiety; and L 
       represents a linker moiety. 
     
     
         9 . A method for the manufacture of a labelled substrate, comprising reacting a compound of general formula III:
   A-L-F  III
   in which A, F and L are as defined in  claim 8 ;   
       with a substrate to form a labelled substrate, 
       wherein the substrate is selected from amino acids, nucleotides, nucleosides, sugars, peptides, proteins, oligonucleotides, polynucleotides, carbohydrates, microparticles and nanoparticles. 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . A functionalised labelling compound having the general formula III:
   A-L-F  III
   
       in which
 A represents a labelling moiety of general formula Ia: 
 
       
         
           
           
               
               
           
         
       
       wherein:
 Fc is a substituted ferrocenyl moiety having at least one ring substituent selected from sulfur-containing groups, phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl groups containing two or more fluorine atoms, heteroaryl, substituted phenyl, and cyano, wherein if present as sole substituent the cyano group is located on the proximal cyclopentadienyl ring; 
 Fc′ is a substituted ferrocenyl moiety having at least one ring substituent selected from sulfur-containing groups, phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl groups containing two or more fluorine atoms, heteroaryl, substituted phenyl, and cyano, wherein if present as sole substituent the cyano group is located on the proximal cyclopentadienyl ring, and may be the same as or different from Fc; 
 X is a C1 to C6 alkylene chain which is optionally interrupted by —O—, —S—, or —NR 5  in which R 5  represents hydrogen or C1 to C6 alkyl; 
 Y is a C1 to C6 alkylene chain which is optionally interrupted by —O— S —, or —NR 5 —, in which R 5  represents hydrogen or C1 to C6 alkyl; 
 Z is a C1 to C12 alkylene chain which may optionally be substituted and/or may optionally be interrupted by —O—, —S—, cycloalkyl, —CO—CONR 1 —, —NR 1 CO— or —NR 1  in which R 1  represents hydrogen or C1 to C4 alkyl; 
 L represents a linker moiety; 
 
       and in which F represents a functionalising moiety for reacting with a substrate for attachment of the labelling moiety to the substrate. 
     
     
         14 . The functionalised labelling compound according to  claim 13 , wherein the substrate is a naturally occurring or synthetic substrate selected from naturally occurring or synthetic amino acids, nucleotides, nucleosides, sugars, peptides, proteins, oligonucleotides, polynucleotides, carbohydrates, microparticles and nanoparticles. 
     
     
         15 . The functionalized labelling compound according to  claim 13 , wherein F is selected from succinimidyl ester groups, phosphoramidite groups, maleimide groups, biotin and azide groups. 
     
     
         16 . The functionalized labeling compound according to  claim 13 , wherein F is or is derivable from a phosphoramidite moiety. 
     
     
         17 . A labelled substrate for use in an electrochemical assay, the labelled substrate being of general formula:
   A-L-F′—[S]  IIIa
   
       in which A represents 
       
         
           
           
               
               
           
         
       
       in which:
 Fc is a substituted ferrocenyl moiety having at least one ring substituent selected from sulfur-containing groups, phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl groups containing two or more fluorine atoms, heteroaryl, substituted phenyl, and cyano, wherein if present as sole substituent the cyano group is located on the proximal cyclopentadienyl ring; 
 Fc′ is a substituted ferrocenyl moiety having at least one ring substituent selected from sulfur-containing groups, phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl groups containing two or more fluorine atoms, heteroaryl, substituted phenyl, and cyano, wherein if present as sole substituent the cyano group is located on the proximal cyclopentadienyl ring, and may be the same as or different from Fc; 
 X is a spacer; 
 Y is a spacer; 
 Z is a spacer; 
 L-F′ represents a linking moiety; and 
 [S] represents a substrate. 
 
     
     
         18 . (canceled) 
     
     
         19 . A labelled substrate according to  claim 17 , wherein the substrate is a microparticle, nanoparticle, biological molecule or a synthetic analog selected from naturally occurring or synthetic amino acids, nucleotides, nucleosides, sugars, peptides, proteins, oligonucleotides, polynucleotides, and carbohydrates. 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . A compound according to general formula I 
       
         
           
           
               
               
           
         
       
       in which:
 Fc is a substituted ferrocenyl moiety having at least one ring substituent selected from sulfur-containing groups, phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl groups containing two or more fluorine atoms, heteroaryl, substituted phenyl, and cyano, wherein if present as sole substituent the cyano group is located on the proximal cyclopentadienyl ring; 
 Fc′ is a substituted ferrocenyl moiety having at least one substituent selected from the group consisting of sulfonyl, having at least one ring substituent selected from sulfur-containing groups, phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl groups containing two or more fluorine atoms, heteroaryl, substituted phenyl, and cyano, wherein if present as sole substituent the cyano group is located on the proximal cyclopentadienyl ring, and may be the same as or different from Fc; 
 X is a spacer; 
 Y is a spacer; 
 Z is a spacer; and 
 R is a linker group. 
 
     
     
         25 . The compound according to  claim 24 , wherein either: (a) X represents C1 to C6-alkylene optionally interrupted by oxygen; Y represents C1 to C6-alkylene optionally interrupted by oxygen, and Z represents C1 to C8 alkylene optionally interrupted by oxygen; or (b) X represents —(CH2) x — in which x is from 1 to 6; and Y represents —(CH2) y — in which y is from 1 to 6. 
     
     
         26 . The compound according to  claim 24 , wherein Z represents C6 to C8 alkylene
 optionally interrupted by oxygen.   
     
     
         27 . The compound according to  claim 24 , in which the linker group R
 comprises a group capable of reacting with a compatible group of a functionalising moiety or of a substrate to attach the compound to said functionalising moiety or said substrate.   
     
     
         28 . (canceled) 
     
     
         29 . The compound of  claim 24 , wherein Fc and Fc′ are the same and each comprise at least one substituent selected from the group consisting of:
 sulfur-containing groups selected from sulfenyl, sulfinyl and sulfonyl groups, 
 phosphorus-containing groups selected from phosphanyl and phosphinyl groups, 
 iodo, 
 chloro, 
 fluoroalkyl groups containing two or more fluorine atoms, 
 heteroaryl, and 
 substituted phenyl. 
 
     
     
         30 . The compound of  claim 24 , wherein Fc and Fc′ are the same and there is present as a said substituent a cyano group located on the respective proximal cyclopentadienyl ring of each of said Fc and Fc′ moieties. 
     
     
         31 . The compound of  claim 24 , wherein Fc and Fc′ are the same and each comprise at least one ring substituent selected from sulfur-containing groups and phosphorus-containing groups. 
     
     
         32 . The compound of  claim 24 , wherein there is present as a said sulfur-containing group at least one sulfonyl substituent selected from groups of the formula R 15 S(0) 2 -, wherein R 15  is selected from branched or straight-chain alkyl, haloalkyl, and substituted or unsubstituted aryl, for example
 wherein R 15  represents C1 to C4 alkyl; or C1 to C4 haloalkyl, for example C1 to C4 fluoroalkyl, especially trifluoromethyl; or.   R 15  represents (R 16 ) a —Ar—, or (R 16 ) a —HeAr— in which Ar represents aryl; HeAr represents heteroaryl; R 16  is a substituent selected from halo, alkyl, nitro, cyano, haloalkyl, and alkoxy; and a is an integer in the range of from 0 to a number equal to the maximum substitutable ring positions in the aryl or heteroaryl ring.   
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . The compound of  claim 24 , wherein Fc and Fc′ are the same and each comprise at least one substituent selected from groups of the formula (R 17 ) 2 P(0)-, wherein R 17  is independently selected from branched or straight-chain alkyl, haloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, for example
 R 17  represents C1 to C4 alkyl or R 17  represents unsubstituted aryl or unsubstituted heteroaryl or represents (R 18 )b-Ar—, or (R 18 ) b —HeAr— in which Ar is aryl; HeAr is heteroaryl; R 18  is a substituent selected from halo, alkyl, nitro, cyano, haloalkyl, and alkoxy; and b is an integer in the range of from 0 to a number equal to the maximum substitutable ring positions in the aryl or heteroaryl ring; for example wherein Ar represents phenyl and comprises one or more substituents R 18  (which may be the same or different) selected from halo, alkyl, haloalkyl, nitro, cyano, and alkoxy, and sulphur-containing groups. 
 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . The compound of  claim 32 , wherein R 17  represents branched C1 to C4 alkyl, for example t-butyl. 
     
     
         40 . The compound of  claim 24 , wherein there is present as a said substituent on each ferrocenyl at least one substituted phenyl group in which the phenyl has at least one substituent selected from halo, C1 to C4 alkyl, nitro, cyano, C1 to C4 haloalkyl, C1 to C4 alkoxy and sulphur-containing groups, for example sulfinyl, sulfenyl and sulfonyl. 
     
     
         41 . The compound of  claim 24 , wherein there is present as a said substituent on each ferrocenyl at least one heteroaryl group which may be unsubstituted or substituted by at least one substituent selected from halo, C1 to C4 alkyl, nitro, cyano, C1 to C4 haloalkyl and C1 to C4 alkoxy, for example wherein the heteroaryl group is furanyl. 
     
     
         42 . (canceled) 
     
     
         43 . The compound of  claim 24 , wherein there is present as a said substituent on each ferrocenyl at least one iodine or chlorine atom. 
     
     
         44 . The compound of  claim 24 , wherein Fc and Fc′ are the same and each comprise as a said substituent group at least one silyl substituent, preferably a silyl group selected from alkyl silyl groups, for example trimethylsilyl. 
     
     
         45 . The compound of  claim 24 , wherein each ferrocenyl moiety is further substituted by at least one additional substituent, for example at least one additional substituent selected from bromo, fluoro, C1 to C4 alkyl, haloalkyl, and C1 to C4 alkenyl. 
     
     
         46 . (canceled) 
     
     
         47 . The compound of  claim 24 , wherein Fc and Fc′ each additionally comprises at least one cyano group substituent on its distal ring. 
     
     
         48 . The compound of  claim 24  wherein the compound is selected from
 6-(bis((2-tert-butyl-sulfonylferrocenyl)1-methylferrocenyl)amino)hexan-1-ol 
 6-(bis((2-cyanoferrocenyl) 1-methylferrocenyl)amino)hexan-1-ol 
 6-(bis((2-di-tert-butyl-phosphinyl-ferrocenyl)1-methylferrocenyl)amino)hexan-1-ol 
 6-(bis((1′-iodoferrocenyl)1-methylferrocenyl)amino)hexan-1-ol 
 6-(bis((1′-(4-nitrobenzyl)ferrocenyl)1-methylferrocenyl)amino)hexan-1-ol 
 6-(bis((1′-(1-furanyl)ferrocenyl) 1-methylferrocenyl)amino)hexan-1-ol 
 6-(bis((1′-chloroferrocenyl) 1-methylferrocenyl)amino)hexan-1-ol 
 6-(bis((2-tert-butyl-sulfinylferrocenyl)1-methylferrocenyl)amino)hexan-1-ol 6-(bis((2-tert-butyl-sulfidylferrocenyl)1-methylferrocenyl)amino)hexan-1-ol. 
 
     
     
         49 . The compound of  claim 24  wherein the compound is a functionalised derivative of a compound according to  claim 48 . 
     
     
         50 . The compound of  claim 24 , wherein R represents a linker group comprising an oxygen atom, for example a linker group comprising a hydroxyl group or a protected hydroxyl group. 
     
     
         51 . A method of detecting a nucleic acid, comprising: (i) contacting the nucleic acid with a complementary nucleic acid probe under conditions to allow hybridization between the probe and amplicon, (i) selectively degrading the either hybridized or unhybridized probe, wherein said probe is labelled with compound according to  claim 24 ; and (iii) measuring the electrochemical activity of the compound labelling the probe, wherein said electrochemical activity is dependent either quantitatively or qualitatively on the extent of degradation of the probe. 
     
     
         52 . (canceled) 
     
     
         53 . (canceled) 
     
     
         54 . A nucleic acid labelled with a compound of  claim 24 .